We have shown that RGS proteins modulate signaling through a variety of G-protein coupled receptors including chemokine receptors. Chemokine receptors signal predominantly by triggering Gi nucleotide exchange. Humans and mice have three Gi isoforms although Gi2 (encoded by Gnai2) and Gi3 (encoded by Gnai3) predominate in lymphoid cells. We have found that Gnai2 -/- T and B cells have severe defects in chemokine-receptor signaling while Gnai2 +/- T and B cells exhibit modest defects. In vivo, the Gnai2-/- B cells fail to properly access lymph node follicles and the Gnai2-/- T cells failing to properly enter the T cell zone. We have studies the immune system with a focus on immune cell trafficking in various RGS deficient mice. We have supplemented these studies with in vitro studies using mRNA knock-downs as well as over expression studies using RGS proteins fused to a fluorescent marker. The recognition of the importance of both G-protein signaling and RGS proteins in the regulation of lymphocyte responses to chemokines has led us to study other hematopoietic cell types. To test the role of Gai2 and Gnai3 in T cell development we examined Gnai3 deficient mice; mice that deleted Gnai2 in CD4 positive cells; and mice that lacked Gnai3 expression and deleted Gnai2 in CD4 positive cells. The loss of Gnai3 had no phenotype, the loss of Gnai2 in CD4 expressing cells led to a mild thymocyte retention phenotype and a reduction in peripheral T cells. Peripheral T cell migration to chemokines was reduced by 40%. The loss of both Gnai2 and Gnai3 in CD4 positive cells led to a severe thymocyte retention phenotype. However, some thymocytes escaped and accumulated within the spleen as mature T cells. These cells exhibited a memory phenotype and most expressed PD-1. Approximately half of the PD-1 positive CD4 T cells expressed CXCR5. Despite their CXCR5 expression they were incapable of migrating to CXCL13 or to any other chemokine. Because of difficulties in generating these mice we performed bone marrow transfers using wild type mice as recipients. These mice had a similar phenotype, but had much more profound peripheral T cell lymphopenia. This suggested that the expansion of PD-1 positive CD4 T cell population that we had observed previously was suppressed by the recipient mouse environment. To test whether radio-resistant T cells in the recipient accounted for the suppressive activity transferred bone marrow into Rag-deficient mice. These mice developed a lymphoproliferative disease with a marked expansion of CD4 positive and PD-1 positive cells that variably express CXCR5. As previously the CD4 T cells in these mice were incapable of migrating to chemokines. As another approach to assessing the role of RGS proteins we have examined mice with a Gnai2 knock-in obtained from Richard Neubig (University of Michigan). This mutation renders Gi2 resistant to the effect of RGS proteins. These KI mice (G194S) possess a striking phenotype verifying the overall important of RGS proteins in G-protein regulation. They have a marked reduction in peripheral T cells; an increase in mature T cells in the thymus; an absence of many peripheral lymph nodes; disorganized lymphoid organ architecture; splenomegaly; abnormal lymphocyte responses to chemokines; and reduced serum levels of IgG3, but increased levels of Ig2b. Neutrophils from these mice accumulate in the bone marrow and mobilize poorly to inflammatory sites. These defects are attributable to enhanced sensitivity to background signals, prolonged chemoattractant receptor signaling, and inappropriate CXCR2 downregulation. Intravital imaging revealed a failure of the mutant neutrophils to accumulate at and stabilize sites of sterile inflammation. Furthermore, these mice could not control a non-lethal Staphylococcus aureus infection (MCB 32:4561-71, 2015). Further analysis of B cells from these mice revealed have they markedly elevated basal calcium levels, but poor chemokine induced increases; enhanced non-specific migration, but extremely poor chemotaxis. In striking contrast, the same B cells exhibited enhanced sensitivity to Sphingosine 1-Phosphate (S1P). Mice with the Gi2 mutation displayed excessive numbers of distorted germinal centers; abnormal serum immunoglobulin profiles; and aberrant B lymphocyte trafficking. These findings established an essential role for RGS proteins in B cell chemoattractant signaling and for the proper position of B lymphocytes in lymphoid organs (J Immunol 194: 2128-39, 2015). Analysis of T cells from these mice revealed that RGS/Gi2 interactions are essential for normal thymocyte egress, T cell trafficking, and homeostasis. Mature KI thymocytes accumulated in the perivascular channel of thymic corticomedullary venules. In the periphery, a severe reduction in nave CD4 T cells and Tregs occurred. Chemokines elicited substandard responses and the KI T cells entered into lymph nodes poorly, yet S1P triggered normal chemotaxis. This study identified a RGS protein dependent step needed for thymocyte reverse transmigration, and illuminate the importance of RGS proteins in T cell trafficking and function. This last study has been submitted for publication.